Connective tissue cells secrete protease inhibitors which are specific for serine proteases. Since serine proteases are involved in development and migration of cells, regulation of the activity of these enzymes is necessary to exercise control over the remodeling or destruction of tissues (Proteases in Biological Control (1975), Reich, E., et al, eds., Cold Spring Harbor, N.Y.). The inhibitors designated protease nexins irreversibly bind to serine proteases at their catalytic sites (Baker, J.B., et al, Cell (1980) 21:37-45) and effect the clearance of the bound proteases via receptor-mediated endocytosis and lysosomal degradation (Low, D. A., et al, Proc Natl Acad Sci (USA) (1981) 78:2340-2344; Baker, J. B., et al, in The Receptors 3 (1985), Conn, P. M., ed, Academic Press, in press).
Three protease nexins have been identified. Protease nexin I (PN-I) has been purified from serum-free medium conditioned by human foreskin cells (Scott, R. W., et al, J Biol Chem (1983) 58:10439-10444). It is a 43 kd glycoprotein which is released by fibroblasts, myotubes, heart muscle cells, and vascular smooth muscle cells. Its release, along with that of plasminogen activator, is stimulated by phorbol esters and by mitogens (Eaton, D. L., et al, J Cell Biol (1983) 123:128). Native PN-I is an approximately 400 amino acid protein containing about 6% carbohydrate. Since it is present only in trace levels in serum, it apparently functions at or near the surfaces of interstitial cells. PN-I inhibits all the known activators of urokinase proenzyme, plasmin, trypsin, thrombin, and factor Xa (Eaton, D. L., et al, J Biol Chem (1984) 259:6241). It also inhibits tissue plasminogen activator and urokinase.
The need for practical amounts of purified PN-I is severalfold. First, PN-I has clear utility as a pharmaceutical for conditions characterized by excess amounts of urokinase and tissue plasminogen activator, or as an antidote for overdoses of these enzymes as agents for solution of blood clots. Indications which are clearly susceptible to PN-I treatment include the autoimmune disease penphigus, which is an extremely common disease in dogs, and psoriasis, which is believed to be due to an overproduction of plasminogen activator. Second, because the role of PN-I in regulating various developmental stages of tissue formation and remodeling is relatively complex, it would be desirable to be able to use model systems to demonstrate in greater detail the role PN-I plays. This can be done effectively only if practical quantities are available. Finally, PN-I is useful as an assay reagent in immunological assays for its levels in serum or in other tissues or for other biological assays.
Exemplary of the conditions for which further study of the role of PN-I is desirable are tumor metastasis, wound healing, and inflammation. In tumor metastasis, malignant cells must penetrate the extracellular matrix laid down by vascular smooth muscle cells, a process which is mediated by secreted plasminogen activator. In the model system of Jones, P. A., et al, Cancer Res (1980) 40:3222, an in vitro system based on the invasion of the extracellular matrix by human fibrosarcoma cells, it could be shown that PN-I at 0.1 .mu.M causes virtually complete suppression of the invasion (Bergman, B. L., et al, Proc Natl Acad Sci USA (1986) 83:996-1000. The proteolytic activity of thrombin, which is a fibroblast mitogen important in wound healing, is effective only when added to cultures at concentrations above the concentrations of secreted PN-I (Baker, J. B., et al, J Cell Physiol (1982) 112:291; Low, D. A., et al, Nature (1982) 298:2476). It has been suggested that PN-I has an antiinflammatory function, since PN-I secretion by synovial fibroblasts increases dramatically when the cells are treated with interleukin-I (McGuire-Goldring et al., Arth Rheum (1984) 27:S24). PN-I may also have a neurological function, since a similar inhibitor which stimulates neurite extension has been shown to be secreted from glial cells (Monard et al, Prog Brain Res (1983) 58:359).
Elucidation of the precise function of PN-I in any of the foregoing would be greatly simplified by the availability of the needed amounts of pure material. These amounts are also needed for use in PN-I as a pharmaceutical and in diagnosis and assay. The present invention provides a solution to the problem of obtaining sufficient quantities of PN-I, as well as a mechanism for modifying PN-I structure in order to make it more effective.